Natural sesquiterpen lactones as acetylcholinesterase inhibitors

Anais da Academia Brasileira de Ciências (2014) 86(2): 801-805 (Annals of the Brazilian Academy of Sciences) Printed version ISSN 0001-3765 / Online version ISSN 1678-2690 http://dx.doi.org/10.1590/0001-3765201420130005 www.scielo.br/aabc Natural sesquiterpen lactones as acetylcholinesterase inhibitors HOMA HAJIMEHDIPOOR1, MAHMOUD MOSADDEGH1,2, FARZANEH NAGHIBI1,2, ALI HAERI3 and MARYAM HAMZELOO-MOGHADAM2 1 Department of Traditional Pharmacy, School of Traditional Medicine, Shahid Beheshti University of Medical Sciences, 1516745811, Tehran, Iran 2 Traditional Medicine and Materia Medica Research Center, Shahid Beheshti University of Medical Sciences, 1516745811, Tehran, Iran, 3 Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, 1516745811, Tehran, Iran Manuscript received on January 7, 2013; accepted for publication on October 14, 2013 ABSTRACT Background and the purpose of the study: The amount of elder people who suffer from Alzheimer disease is continuously increasing every year. Cholinesterase inhibitors have shown to be effective in alleviating the symptoms of the disease, thus opening a field of research for these treatments. Herbal products, owning a reputation as effective agents in many biological studies are now drawing attention for inhibiting acetyl cholinesterase, in other words, Alzheimer disease. In the present study, the ability of three sesquiterpene lactones from Inula oculus-christi and I. aucheriana to inhibit AChE has been evaluated through Ellman assay. Materials and Methods: Gaillardin and pulchellin C were obtained from I. oculus-christi and britannin from I. aucheriana by chromatographic methods. They were dissolved in methanol in concentration of 3 mg/mL and the AChEI activity of the compounds was determined by Ellman method using Acethylthiocholine iodide as the substrate and 5, 5'-dithiobis-2-nitrobenzoic acid as the reagent, in 96-well plates at 405 nm. Results: AChEI activity of the examined compounds was obtained as 67.0, 25.2 and 10.9% in concentration of 300 μg/L for gaillardin, britannin and pulchellin C, respectively. Conclusion: Among the three sesquiterpene lactones, gaillardin with 67% inhibition of AChE could be considered a good candidate for future Alzheimer studies. Key words: Acetylcholinesterase inhibitor, Alzheimer, britannin, gaillardin, Inula, pulchellin C. INTRODUCTION The type of dementia that elder people often suffer, is called Alzheimer disease (AD) and influences the life of more than 25 million a year (D’Addario et al. 2012). The number is rapidly growing, which clearly determines the importance of the need for a careful diagnosis and the handling of the illness (Farlow and Lilly 2005). Commonly, AD begins Correspondence to: Maryam Hamzeloo-Moghadam E-mail: with a gradual worsening of the ability to remember new information. As damage expands, other difficulties occur, such as memory loss, confusion with time or place, misplacing things and losing the ability to retrace steps as well as a decreased or poor judgment. The cause of AD is still unknown but like other common chronic conditions, it progresses as a result of several factors and a variety of brain changes that begin as many as 20 years before symptoms show up (Alzheimer’s Association 2012). Based on An Acad Bras Cienc (2014) 86 (2) 802 HOMA HAJIMEHDIPOOR et al. the cholinergic hypothesis, acetylcholinesterase inhibitors (AChEIs) are widely used to alleviate AD. Nowadays investigations to find AChEI materials from natural sources have increased and it has been proven that many compounds of different structures have AChEI property; among which alkaloids are the most ruling compounds. Agents such as the alkaloid galanthamine, are examples possessing the mentioned property (Feitosa et al. 2011).There are not as many reports about AChEI activity of sesquiterpenes as alkaloids, but the existing data are indicative of the mentioned activity in some herbs and their sesquiterpene constituents. The results of isolating sesquiterpene lactones from Amberboa ramosa, a plant of Asteraceae, have revealed their excellent AChEI activity (Ibrahim et al. 2013); while the leaves essential oil of Hedychium gardnerianum has exhibited AChEI activity (Arruda et al. 2012) recommending the oil to be used in aromatherapy. Since the constituents were mostly found to be sesquiterpenes, the authors had concluded that the AChEI activity was due to the presence of these compounds. The n-hexane fractions of the peels and leaves of bitter orange have also demonstrated to be inhibitors of AChE and gas chromatography-mass spectrometry analysis have again deduced the most dominant compounds to be mono- and sesquiterpenes (Loizzo et al. 2012). These results attracted our attention to the possibility of other sesquiterpenes to encompass the ability to inhibit AChE and in the present study, sesquiterpene lactones isolated from two species of Inula (Asteraceae) named I. oculus-christi L. and I. aucheriana DC. have been investigated through Ellman assay. MATERIALS AND METHODS CHEMICALS Acetylcholinesterase (AChE) was purchased from Sigma, Germany. Acetylthiocholin Iodide (ATCI) was prepared from Fluka, Germany. 5, 5'-dithiobis2-nitrobenzoic acid (DTNB) and other solvents and chemicals were provided from Merck, Germany. An Acad Bras Cienc (2014) 86 (2) PLANT MATERIAL In June 2009, Inula oculus-christi L. was collected from Golestan province and Inula aucheriana DC. was collected from West Azerbaijan province in Iran (July 2010). The species were authenticated by Mrs. Atefeh Pirani (Botanist), Traditional Medicine and Materia Medica Research Center (TMRC), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Voucher specimens of both species have been deposited at TMRC Herbarium for future reference. The aerial parts of the plants were dried in shade and ground. EXTRACTION AND ISOLATION Gaillardin and pulchellin C were obtained from I. oculus-christi (Mosaddegh et al. 2010); briefly, the chloroform extract of 250 g of the aerial parts was further subjected to column chromatography to afford gaillardin and pulchellin C. Britannin was obtained from chloroform fraction of 500 g I. aucheriana, through serial extraction with n-hexane, chloroform and methanol (HamzelooMoghadam et al. 2012). The chemical structures of the compounds are presented in figure 1. ACETYLCHOLINESTERASE INHIBITORY ACTIVITY ASSAY Ellman assay is usually applied in vitro to define the cholinesterase activity of materials. It was established by Ellman et al. (1961) and is based on the reaction of thiocholine (one of the products of enzymatic hydrolysis of acetylthiocholine) with DTNB (Ellman’s reagent), forming a yellow product (5-mercapto- 2-nitrobenzoic acid and its dissociated forms) at pH 8 which can be detected at 405 nm (Komersova et al. 2007). Based on the above mentioned assay, the experiment was con ducted according to modified microplate method (Mukherjee (...truncated)